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CASE STUDY

1

Structural stabilization due to monoclonal antibody binding influences the protease activity of human kallikrein-3

• Accurateprotein-proteincomplexstructurepredictionandrefinementwithZDOCKandRDOCKprotocols

• CHARMmbasedCalculateMutationEnergyprotocolpredictsantibodymutantswithenhancedstability

Structural characterization of an antibody in complex with a Serine proteaSe

Proteases are widely distributed in nature

and various deregulated protease activities

are frequently implicated in pathological

conditions such as cancer, cardiovascular

and inflammatory disorders. The challenge

in developing an inhibitor for a particular

protease is to achieve specificity, since

members of the same protease family

often have largely conserved binding sites.

The highly specific nature of antibody-

antigen recognition makes it desirable

to consider designing antibodies as

potential modifiers of protease functions.

Prostate-specific Antigen (PSA), also known

as kallikrein-3, is an androgen-regulated

serine protease expressed in prostate

tissue and shares the characteristic His-

Asp-Ser catalytic triad of serine proteases.

PSA is a well known biomarker for prostate

cancer, and implicated in prostate cancer

development and progression. However

the exact role of its protease activity in

prostate cancer is still unknown. Recently the

crystal structure of kallikrein-3 in complex

with an activating antibody has been

solved (Menez et al, 2008), which affords

insight into the structural determinants

of recognition and sheds light on the

nature of antibody-PSA interactions.

Sincetheadventofmonoclonalantibody(mAb)technologiesinrecentyears,

numeroustherapeuticmAbshavebeendevelopedintobeneficialagentsforthe

treatmentofavarietyofhumandiseases.Antibodiesaremulti-domainstructures

typicallycomposedoftwolightchainsandtwoheavychains.Thevariabledomain

ofeachchaincontainsthreeregionsofsequencehypervariability,calledtheCDRs

(complementarity-determiningregions).Theseversatilehypervariableregionsare

responsiblefortheantigenbindingaffinityandspecificity.

Tina Yeh, PhD Lead Scientist - Accelrys

CASE STUDY: DiSCovErY STUDio

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method

Protein docking studies were performed in Discovery Studio

using the Dock Proteins protocols. The ZDOCK protocol performs

the initial global, systematic search of the orientations of the two

protein partners. Typically the larger protein (the receptor protein)

is kept fixed while moving the smaller protein (the ligand protein)

around the receptor protein. ZDOCK uses a grid-based rigid-

body docking search in six dimensions utilizing the Fast Fourier

Transform (FFT) technique for efficiency. The rotational search

sampling grid can use a 15 degree grid which samples a total

of 3600 docked poses, or a 6 degree grid which samples a total

of 54,000 poses for more accurate results. The ZDOCK internal

scoring algorithm is based on a pairwise shape complementarity

function (PSC) and optionally delsolvation and electrostatic

terms can also be included. As part of the ZDOCK protocol, the

ZRANK function is used to rerank the docked poses. ZRANK is an

optimized energy scoring function based on weighted energy

terms of van der Waals, electrostatics, and desolvation. The RDOCK

protocol can be used subsequently for further refinement of the

docked poses, using a CHARMm-based energy minimization

scheme for the optimization of intermolecular interactions.

PSA and the antibody Fab 8G8F5 structures are taken from PDB

2ZCH. The ZDOCK protocol with a 6 degree rotational sampling

grid was used and several antibody CDR loop residues were

specified for filtering poses. Docked poses are ranked with ZRANK

and further optimized with RDOCK. The best pose is very similar

to the crystal structure of 2ZCH (Figure 1), demonstrating that

the ZRANK scoring function and RDOCK refinement successfully

discriminate the near native protein complex structure.

The antibody 8G8F5 recognizes the binding epitope on the

antigen PSA located at five discontinuous surface loop segments

including residues: 90-95, 98-101, 124-129, 175-179, 232-240

(see Figure 2). The antibody does not reach into the PSA active

site; rather it binds at the peripheral side of the active site cleft.

The heavy chain CDR loops contribute more to the binding

interaction than do the light chain CDR loops. All 3 heavy

chain CDR loop residues H:27-33, H:52-58, H:96-100C are in the

central core region of the binding interface whereas the light

chain CDR1 loop residues L:27D-32 and CDR3 loop residues

L:91-96 residues are involved in the peripheral binding area.

As shown in the figure above, the three PSA loops [90-95] [98-101]

[175-179] are all in close spatial proximity and interact with all

three of the antibody’s heavy chain CDRs. The so-called classic

“Kallikrein loop” in PSA includes an 11 amino acids insertion

95A-95K (relative to standard chymotrypsin numbering). The

kallikrein loop in PSA is located between loop [90-95] and

loop [98-101], at the border of the PSA active site cleft.

PSA:His91 forms a hydrogen bond with antibody heavy

chain CDR3 residue H:Tyr97, and PSA:Pro92 forms a hydrogen

bond with heavy chain CDR2 residue H:Ser54. PSA:Leu93

is located in a hydrophobic pocket with favorable van der

Waals interaction with surrounding antibody heavy chain CDR

residues including H:Tyr33, H:Ala52, H:Pro52A, and H:Tyr97.

figure 1. Protein ribbon diagram of the best docking prediction of the PSA-antibody complex superimposed on the crystal structure from PDB 2ZCH. Antibody light chain (orange), heavy chain (green).

figure 2. Two views of the PSA-antibody binding interface are shown as ribbon diagrams. Antibody heavy chain (green) and light chain (orange). The antigenic determinants on PSA include five discontinuous segments: three loops (purple) [90-95] [98-101] [175-179], one loop [124-129] (yellow) and the C-terminal helical region [232-240] (yellow). The PSA catalytic triad residues H57 D102 S195 are labeled.

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The PSA:Asp98 side chain can form hydrogen bonds with

antibody residue H:Thr28 or H:Thr31. At one edge of the

binding interface the PSA loop residue PSA:Lys175 also forms

a hydrogen bond with antibody residue H:Thr28. Another

PSA lysine residue PSA:Lys178 is within hydrogen bond

distance with antibody residues H:Asp96 and H:Tyr97.

The last segment PSA:232-240 is part of the PSA C-terminal helix

and interacts with the CDR loops from both the antibody light

chain and the heavy chain. At the edge of the binding interface

PSA:Lys236 can form a hydrogen bond with antibody residue

L:Ser91 and a salt bridge interaction with H:Glu100C. PSA:Arg235

forms a hydrogen bond with antibody residue L:Asp28.

With the solvent surface displayed on PSA, it’s easy to visualize the

different areas where each antibody CDR loop residue interacts

with PSA (see Figure 3). The heavy chain CDR3 loop occupies the

core region of the binding interface. The side chain of the two

lysine residues on PSA, Lys178 and Lys236, delineate a concave

shaped pocket on PSA that the heavy chain CDR3 loop fits

into. Another pair of lysine residues on PSA, Lys178 and Lys175,

delineate another pocket that the heavy chain CDR1 loop fits into.

At the edge of the binding interface, PSA residue Lys239 interacts

favorably with antibody light chain CDR1 loop residue L:Phe27D.

Several hydrophobic and aromatic amino acids from the

antibody heavy chain CDR3 loop and from PSA contribute to

the stabilizing hydrophobic core and favorable π interactions

in the binding interface. These include residues: H:Tyr32,

H:Tyr33, H:Tyr58, H:Tyr97, H:Phe99, and PSA:Leu93, PSA:His91,

PSA:His101, PSA:Phe179, PSA:Tyr234, PSA:Trp237.

Since PSA, also known as kallikrein-3, has a very long kallikrein

loop including the 11 residue insertion (from Met95A to Pro95K),

the flexible loop conformation could block the access to the PSA

active site nearby. Based on the allosteric regulation (activation)

mechanism, it has been proposed (Menez et al, 2008) that, the

antibody interacts with the two PSA surface loops [90-95] and

[98-101] on either side of the kallikrein loop, thereby effectively

stabilizing the kallikrein loop conformation and keeping the

substrate-binding cleft accessible. This could explain the

enhanced PSA enzyme activity upon antibody binding.

We have performed computational scanning mutagenesis

analysis using the CHARMm-based Calculate Mutation Energy

protocols in Discovery Studio, to evaluate the effect of single-

point mutations on the stability and binding affinity of the

PSA-antibody complex. For the Mutation Energy (Stability)

calculation, the energy effect of each mutation is calculated as

the difference between the folding free energy of the mutated

structure and the wild type structure. The method includes the

Generalized Born implicit solvent model in CHARMm and the

energy functional contains empirically scaled contributions

of van der Waals and electrostatic terms, a side chain entropy

term and a non-polar solvation energy term. The results have

identified several mutations on the antibody heavy chain that

exhibit a stabilizing effect. These include residue H:Ser54 on

the heavy chain CDR2 loop mutated to hydrophobic amino

acids TRP, TYR, PHE, ILE, and LEU, all stabilizing mutations, with

favorable contribution from the van der Waals energy term.

Residue H:Thr31 on the heavy chain CDR1 loop mutated

to MET, ILE, PHE, and VAL are also stabilizing mutations.

figure 3. Four lysine residues involved in the binding interface. PSA is represented as a surface with the four lysine residues (Blue) labeled. Ribbon diagram for the three heavy chain CDR loops displayed in yellow, with CDR3 loop in the center of the screen (between Lys236 and Lys178). Ribbon diagram for the light chain CDR1 and CDR3 loops displayed in red.

CASE STUDY: DiSCovErY STUDio

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concluSion

In this case study we demonstrate the structural analysis

of the binding interface of a PSA-antibody complex

using the tools and methods in Discovery Studio.

Protein docking studies using ZDOCK and RDOCK refinement

has successfully predicted the near native structure for the

PSA-antibody complex as determined by X-ray crystallography.

All three CDR loops of the antibody heavy chain contribute

the major part of the interaction with PSA, with the CDR3

loop at the core of the binding interface. The antibody light

chain CDR1 and CDR3 loops provide interaction at the edge

of the binding interface. The antibody-PSA binding affinity is

achieved through the shape complementarity between the

antibody paratope and the epitope on PSA consisting of five

discontinuous segments, the extensive hydrogen bonds, and

favorable van der Waals and hydrophobic interactions. The

computational scanning mutagenesis study has identified

a few putative mutations on the antibody heavy chain that

could potentially enhance the stability of the complex.

To learn more about Discovery Studio by Accelrys, go to

accelrys.com/discovery-studio

referenceS

1. Menez et al, “Crystal Structure of a Ternary Complex between Human

Prostate-specific Antigen, Its Substrate Acyl Intermediate and an

Activating Antibody,” J. Mol. Biol. 2008, 376, 1021-1033.

2. LeBeau et al, “Prostate-specific antigen: an overlooked candidate for

targeted treatment and selective imaging of prostate cancer,” Biol. Chem.

2010, 391, 333-343

3. Wu et al, “Structural insight into distinct mechanisms of protease inhibition

by antibodies,” PNAS 2007, 104(50), 19784-19789

4. Hedstrom L. “Serine Protease Mechanism and Specificity,” Chem. Rev. 2002,

102, 4501-4523.

5. Pierce B., Weng Z. “A Combination of Rescoring and Refinement

Significantly Improves Protein Docking Performance,” Proteins 2008,

72, 270-279.